Incompatibility Group IncI1 Research Articles

Molecular Characterization of Escherichia coli Producing Extended-Spectrum ß-Lactamase and MCR-1 from Sick Pigs in a Greek Slaughterhouse.

The first prospective surveillance of ESBL and colistin-resistant Escherichia coli recovered from sick pigs from a slaughterhouse in Central Greece aimed to investigate the spread of relevant genetic elements. In February 2021, 25 E. coli isolates were subjected to antimicrobial susceptibility testing using disk diffusion and broth microdilution techniques. PCR screening was conducted to identify ESBLs and mcr genes. Additional assays, encompassing mating-out procedures, molecular typing utilizing Pulsed-Field Gel Electrophoresis, multilocus sequence typing analysis, and plasmid typing, were also conducted. A 40% prevalence of ESBLs and an 80% prevalence of MCR-1 were identified, with a co-occurrence rate of 32%. The predominant ESBL identified was CTX-M-3, followed by SHV-12. Resistance to colistin, chloramphenicol, cotrimoxazol, and ciprofloxacin was detected in twenty (80%), fifteen (60%), twelve (48%), and four (16%) isolates, respectively. All blaCTX-M-3 harboring plasmids were conjugative, belonging to the incompatibility group IncI1, and approximately 50 kb in size. Those carrying blaSHV-12 were also conjugative, classified into incompatibility group IncI2, and approximately 70 kb in size. The mcr-1 genes were predominantly located on conjugative plasmids associated with the IncX4 incompatibility group. Molecular typing of the ten concurrent ESBL and MCR-1 producers revealed seven multilocus sequence types. The heterogeneous population of E. coli isolates carrying resistant genes on constant plasmids implies that the dissemination of resistance genes is likely facilitated by horizontal plasmid transfer.

Incompatibility Group I1 (IncI1) Plasmids: Their Genetics, Biology, and Public Health Relevance.

Bacterial plasmids are extrachromosomal genetic elements that often carry antimicrobial resistance (AMR) genes and genes encoding increased virulence and can be transmissible among bacteria by conjugation. One key group of plasmids is the incompatibility group I1 (IncI1) plasmids, which have been isolated from multiple Enterobacteriaceae of food animal origin and clinically ill human patients. The IncI group of plasmids were initially characterized due to their sensitivity to the filamentous bacteriophage If1. Two prototypical IncI1 plasmids, R64 and pColIb-P9, have been extensively studied, and the plasmids consist of unique regions associated with plasmid replication, plasmid stability/maintenance, transfer machinery apparatus, single-stranded DNA transfer, and antimicrobial resistance. IncI1 plasmids are somewhat unique in that they encode two types of sex pili, a thick, rigid pilus necessary for mating and a thin, flexible pilus that helps stabilize bacteria for plasmid transfer in liquid environments. A key public health concern with IncI1 plasmids is their ability to carry antimicrobial resistance genes, including those associated with critically important antimicrobials used to treat severe cases of enteric infections, including the third-generation cephalosporins. Because of the potential importance of these plasmids, this review focuses on the distribution of the plasmids, their phenotypic characteristics associated with antimicrobial resistance and virulence, and their replication, maintenance, and transfer.

Colistin-Resistant Enterobacteriaceae Isolated From Process Waters and Wastewater From German Poultry and Pig Slaughterhouses.

Due to the high prevalence of colistin-resistant Enterobacteriaceae in poultry and pigs, process waters and wastewater from slaughterhouses were considered as a hotspot for isolates carrying plasmid-encoded, mobilizable colistin resistances (mcr genes). Thus, questions on the effectiveness of wastewater treatment in in-house and municipal wastewater treatment plants (WWTPs) as well as on the diversity of the prevailing isolates, plasmid types, and their transmissibility arise. Process waters and wastewater accruing in the delivery and unclean areas of two poultry and two pig slaughterhouses were screened for the presence of target colistin-resistant bacteria (i.e., Escherichia coli, Klebsiella spp., Enterobacter cloacae complex). In-house and municipal WWTPs (mWWTPs) including receiving waterbodies were investigated as well. Samples taken in the poultry slaughterhouses yielded the highest occurrence of target colistin-resistant Enterobacteriaceae (40.2%, 33/82), followed by mWWTPs (25.0%, 9/36) and pig slaughterhouses (14.9%, 10/67). Recovered isolates exhibited various resistance patterns. The resistance rates using epidemiological cut-off values were higher in comparison to those obtained with clinical breakpoints. Noteworthy, MCR-1-producing Klebsiella pneumoniae and E. coli were detected in scalding waters and preflooders of mWWTPs. A total of 70.8% (46/65) of E. coli and 20.6% (7/34) of K. pneumoniae isolates carried mcr-1 on a variety of transferable plasmids with incompatibility groups IncI1, IncHI2, IncX4, IncF, and IncI2 ranging between 30 and 360 kb. The analyzed isolates carrying mcr-1 on transferable plasmids (n = 53) exhibited a broad diversity, as they were assigned to 25 different XbaI profiles. Interestingly, in the majority of colistin-resistant mcr-negative E. coli and K. pneumoniae isolates non-synonymous polymorphisms in pmrAB were detected. Our findings demonstrated high occurrence of colistin-resistant E. coli and K. pneumoniae carrying mcr-1 on transferrable plasmids in poultry and pig slaughterhouses and indicate their dissemination into surface water.

Evaluation of Incompatibility Group I1 (IncI1) Plasmid-Containing Salmonella enterica and Assessment of the Plasmids in Bacteriocin Production and Biofilm Development.

Mobile genetic elements, such as plasmids, can potentially increase the ability of bacteria to infect and persist in vertebrate host cells. IncI1 plasmids are widely distributed in Salmonella from food animal sources and associated with clinically important strains. These plasmids often encode antimicrobial resistance; however, little is known about their impact on the virulence of Salmonella strains. To assess the potential impact of the plasmids on virulence, 43 IncI1-positive Salmonella isolates from human and animal sources were subjected to whole genome sequence (WGS) analyses and evaluated for their abilities to invade and persist for 48 h in Caco-2 human intestinal epithelial cells, form biofilms and encode bacteriocins. Draft WGS data were submitted to predict the presence of virulence and antimicrobial resistance genes, plasmid replicon types present, conduct plasmid multilocus sequence typing (pMLST), and core genome MLST (cgMLST) in the isolates. Caco-2 cells were infected with Salmonella strains and incubated for both one and 48 h for the invasion and persistence assays, respectively. Additionally, Salmonella isolates and IncI1 plasmid carrying transconjugants (n = 12) generated in Escherichia coli were assessed for their ability to produce biofilms and bacteriocin inhibition of growth of other bacteria. All Salmonella isolates infected Caco-2 cells and persisted in the cells at 48 hrs. Persistent cell counts were observed to be significantly higher than invasion assay cell counts in 26% of the isolates. Among the IncI1 plasmids, there were 18 pMLST types. Nearly 35% (n = 15) of Salmonella isolates produced biofilms; however, none of the IncI1-positive transconjugants produced increased biofilms compared to the recipient. Approximately 65% (n = 28) of isolates and 67% (n = 8) of IncI1-positive transconjugants were able to inhibit growth of at least one E. coli strain; however, none inhibited the growth of strains from species other than E. coli. The study characterized IncI1 positive Salmonella isolates and provided evidence about the potential contributions of IncI1 plasmids virulence phenotypes and areas where they do not. These findings should allow for more focused efforts to assess the impact of plasmids on bacterial pathophysiology and human health.

Whole-Genome Sequences of 35 Incompatibility Group I1 Plasmid-Carrying Salmonella enterica Isolates from Food Animal and Clinical Sources.

We sequenced 35 Salmonella enterica isolates carrying incompatibility group I1 (IncI1) plasmids from different serotypes to study their genotypic characteristics. The isolates originated from food animals (n = 32) and human patients (n = 3). All isolates carried IncI1 plasmids, and many had additional plasmids detected along with virulence and antimicrobial resistance genes.

Characterization of Salmonella enterica serovars recovered from meat products legally and illegally imported into the EU reveals the presence of multiresistant and AmpC-producing isolates

BackgroundFood products of animal origin brought into the EU from third countries, both legally and illegally, can harbor foodborne pathogens such as Salmonella enterica. In this study, we examined five S. enterica isolates recovered either from legally imported chicken meat (n = 3) or from meat products confiscated from air travel passengers arriving in Germany (n = 2). The isolates were serotyped and further characterized by antimicrobial susceptibility testing, PCR-detection and sequencing of genes associated with antimicrobial resistances, and macrorestriction analysis. Transferability of resistance to third-generation cephalosporins was assessed by conjugation experiments and the plasmids tested for their incompatibility groups.ResultsThe three isolates from legal imports were identified as S. Heidelberg or as non-flagellated. All three isolates were identified as AmpC producers carrying blaCMY-2 and as non-susceptible to ciprofloxacin. They were additionally resistant to tetracycline and sulfamethoxazole. The blaCMY-2-carrying plasmids were transferable by conjugation and belonged to incompatibility groups IncI1 or IncA/C. The two isolates from illegally imported meat belonged to the serovars Infantis or Weltevreden. The former was phenotypically resistant to five classes of antimicrobial agents while the S. Weltevreden isolate was fully susceptible to all agents tested.ConclusionThe results of this study demonstrate that meat products imported from third countries, both legally and illegally, can harbor multiresistant Salmonella enterica. Consequently, these imports could constitute a source for the dissemination of antimicrobial resistant isolates, including those resistant to third-generation cephalosporins and fluoroquinolones.

Evaluation of the Genetics and Functionality of Plasmids in Incompatibility Group I1-Positive Salmonella enterica.

Salmonella is a predominant foodborne pathogen in the United States and other countries. Mobile genetic elements such as plasmids allow Salmonella to adapt to external stress factors such as nutrient deprivation and host factors. Incompatibility group I1 (IncI1) plasmid-carrying Salmonella enterica strains were examined to determine the presence of plasmid-associated genes and their influence on phenotypic characteristics. The objective of this study was to understand the genetic determinants on IncI1 plasmids and their impact on antimicrobial susceptibility, competitive growth inhibition of Escherichia coli, and plasmid transfer. Primers were designed for genes that play a role in virulence, antimicrobial resistance, and plasmid transfer based on previously sequenced IncI1 plasmids. Polymerase chain reaction assays were conducted on 92 incompatibility group I1 (IncI1)-positive S. enterica strains. Phenotypic characterization included conjugation assays, antimicrobial susceptibility testing, and bacteriocin production based on the inhibition of growth of colicin-negative E. coli J53. The antimicrobial resistance genes aadA1, tetA, sul1, and blaCMY were detected in 88%, 87%, 80%, and 48% of the strains, respectively. Over half of the strains were resistant or intermediately resistant to streptomycin (85%), sulfonamides (76%), tetracycline (74%), and ampicillin (68%) and 57% of the strains inhibited growth of E. coli J53 strain. Among putative virulence genes, colicin-associated colI and cib were detected in 23% and 35% of strains and imm and ccdA were present in 58% and 54% of strains, respectively. Approximately 61% of strains contained plasmids that conjugally transferred antimicrobial resistance, including 83% where the recipient received IncI1 plasmids. Most of the strains carried an assortment of transfer associated (pil and tra) genes with between 63% and 99% of strains being positive for individual genes. Taken together the study affirms that IncI1 plasmids likely play roles in the dissemination of antimicrobial resistance and virulence-associated factors among enteric organisms.

Draft Genome Sequences of Salmonella enterica Isolates Containing Incompatibility Group I1 Plasmids from Swine, Poultry, and Human Sources.

ABSTRACTThe draft genome sequences of eight Salmonella enterica isolates from various sources were evaluated for the influence of incompatibility group I1 (IncI1) plasmids on virulence. Strains SE142, SE143, SE144, and SE146 originated from swine, SE36N and SE89N from poultry-related sources, and SE991 and SE1148 from human patients.

Analysis of blaSHV-12-carrying Escherichia coli clones and plasmids from human, animal and food sources.

This study aimed at characterizing 23 Escherichia coli isolates from various sources and their respective bla SHV-12 -carrying plasmids and sequencing one of these plasmids completely. Isolates were typed by XbaI-PFGE, MLST and PCR-based phylotyping. Transformed bla SHV-12 -carrying plasmids were examined by replicon typing, S1-nuclease, conjugation, EcoRI-HindIII-BamHI digests and plasmid MLST. Co-located resistance genes and integrons as well as the bla SHV-12 genetic environment were analysed by PCR and sequencing. One IncI1 plasmid was sequenced completely using HiSeq 2500 and gap closure by PCRs and Sanger sequencing. Among the 23 SHV-12-positive E. coli , some isolates from different sources showed the same characteristics: ST23/phylogroup A (human, dog, livestock), ST57/D (wild bird, chicken meat) and ST117/D (chicken meat, chicken). All bla SHV-12 genes were horizontally transferable via 30-120 kb plasmids of incompatibility groups IncI1 ( n = 17), IncK ( n = 3), IncF ( n = 1), IncX3 ( n = 1) and a non-typeable plasmid. IncK plasmids, indistinguishable in size and restriction patterns, were found in isolates from different sources (ST57/D, meat; ST131/B2, meat; ST57/B1, dog). The IncI1- bla SHV-12 -carrying plasmids were mostly assigned to plasmid ST (pST) 26 and pST3. Three plasmids showed novel pSTs (pST214, pST215). The majority of the IncI1 transformants exhibited resistance to β-lactams, chloramphenicol and streptomycin (in relation with a class 1 integron containing an estX - psp - aadA2 - cmlA1 - aadA1 - qacI gene cassette array), and to tetracycline. A novel bla SHV-12 environment was detected and whole plasmid sequencing revealed a Tn 21 -derived- bla SHV12 -ΔTn 1721 resistance complex. Results from this study suggest that the dissemination of bla SHV-12 genes occurs by vertical (clonal) and horizontal transfer, the latter mainly mediated through IncI1 multidrug-resistance plasmids.

Characterization of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli obtained from Danish pigs, pig farmers and their families from farms with high or no consumption of third- or fourth-generation cephalosporins.

To compare and characterize extended-spectrum β-lactamase (ESBL)-producing Escherichia coli from pigsties, pig farmers and their families on farms with previous high or no use of third- or fourth-generation cephalosporins. Twenty farms with no third- or fourth-generation cephalosporin use and 19 herds with previous frequent use were included. The ESBL-producing isolates detected in humans and pigs were characterized by ESBL genotype, PFGE, susceptibility to non-β-lactam antibiotics and phylotype, and selected isolates were characterized by multilocus sequence typing (MLST). Furthermore, transferability of bla(CTX-M-)1 from both human and pig isolates was studied and plasmid incompatibility groups were defined. The volunteers answered a questionnaire including epidemiological risk factors for carriage of ESBL-producing E. coli. ESBL-producing E. coli was detected in pigs on 79% of the farms with high consumption of cephalosporins compared with 20% of the pigs on farms with no consumption. ESBL-producing E. coli was detected in 19 of the 195 human participants and all but one had contact with pigs. The genes found in both humans and pigs at the same farms were blaCTX-M-1 (eight farms), bla(CTX-M-14) (one farm) and bla(SHV-12) (one farm). At four farms ESBL-producing E. coli isolates with the same CTX-M enzyme, phylotype, PFGE type and MLST type were detected in both pigs and farmers. The majority of the plasmids with bla(CTX-M-1) were transferable by conjugation and belonged to incompatibility group IncI1, IncF, or IncN. The present study shows an increased frequency of ESBL-producing E. coli on farms with high consumption of third- or fourth-generation cephalosporins and indicates transfer of either ESBL-producing E. coli or plasmids between pigs and farmers.

Efficient mobilization of a resistance derivative of pSLT, the virulence plasmid specific of Salmonella enterica serovar Typhimurium, by an IncI1 plasmid

pUO-StVR2 is a derivative of pSLT, the virulence plasmid specific of Salmonella enterica serovar Typhimurium, which confers multidrug resistance. This plasmid is widespread among closely related isolates of S. Typhimurium, and often coexists with other plasmids like pStR12. The latter belongs to incompatibility group IncI1, was assigned to ST48 by pMLST (plasmid multilocus sequence typing), and confers resistance to streptomycin/spectinomycin, chloramphenicol, trimethoprim and sulphonamides, with the responsible genes (aadA1/aadA2, cmlA1, dfrA12 and sul3) located on a sul3-class 1 integron. When using clinical isolates of S. Typhimurium containing one (pUO-StVR2) or both (pUO-StVR2 and pStR12) plasmids as donors and Escherichia coli K12 J53 resistant to rifampicin as recipient, the conjugation frequencies of pUO-StVR2 and pStR12 were 10−8 and 10−3–10−5 transconjugants/donor, respectively, while the transfer frequency of pUO-StVR2 increased 102 up to 105 times through mobilization by pStR12, depending on the donor strain and experimental conditions. Mobilization of pUO-StVR2, a plasmid which encodes virulence and resistance functions, by compatible plasmids which coexist in the same bacterium can facilitate the spread of these properties in S. Typhimurium, one of the most common serovars of S. enterica.

The plasmidome of a Salmonella enterica serovar Derby isolated from pork meat

The complete nucleotide sequences of four plasmids hosted by a Salmonella enterica serovar. Derby strain 6MK1 isolated from pork were determined by shotgun Sanger sequencing. A 107,637 base pairs (bp) conjugative plasmid pSD107 containing 150 putative coding sequences (CDS) could be assigned to the narrow host range incompatibility group IncI1. A detailed annotation of all CDS was carried out, revealing the presence of genes needed for plasmid replication, conjugal transfer, plasmid partitioning and stability as well as resistance to antimicrobials. The resistance determinants dhfrA1, aadA1, qacEΔ1, sul1 (supplied by a class 1 integron), blaTEM−1b (carried by a truncated Tn2 flanked by IS26), sul2 and strAB confer multidrug resistance to the host bacterium. In addition to pSD107, three small cryptic plasmids pSD4.0, pSD4.6 and pSD5.6 were identified, showing significant sequence similarities to already known replicons of Escherichia coli and S. enterica. In conjugation experiments performed on solid medium, pSD107 was successfully transferred to a nalidixic acid resistant E. coli DH5α, mobilizing pSD4.0 and, more infrequently, also pSD4.6. All transferred plasmids were stably propagated in the recipient strain without selective pressure for approximately 66 generations. The absolute plasmid copy numbers were determined in real time PCR experiments, revealing an approximate 1:1:1:1 ratio of the four replicons compared to the chromosome. The evolutionary position of pSD107 within the IncI1 family of plasmids was inferred from a maximum likelihood phylogenetic tree and by comparison of genetic key elements in a set of 17 IncI1 reference plasmids.

Complete genome sequence of the incompatibility group I1 plasmid R64

A streptomycin and tetracycline resistance plasmid R64 isolated from Salmonella enterica serovar Typhimurium belongs to the incompatibility group I1 (IncI1). The DNA sequence of the R64 conjugative transfer region was described previously ( Komano et al., 2000). Here, we report the complete genome sequence of R64. In the circular double-stranded R64 genome with 120,826 bp, 126 complete ORFs are predicted. In addition, 2 and 6 different kinds of proteins are produced by translational reinitiation and shufflon multiple inversions, respectively. The genome consists of five major regions: replication, drug resistance, stability, transfer leading, and conjugative transfer regions in clockwise order. The nucleotide sequence essential for autonomous replication of R64 is completely identical to that of IncI1 colicinogenic plasmid ColIb-P9, an indication that these two plasmids share the same mechanisms for replication and copy number control. Tetracycline and streptomycin resistance genes are encoded in transposons Tn 10 and Tn 6082, respectively. These transposons and two insertion elements, IS 2 and IS 1133, were inserted stepwise into the arsenic-resistant gene, arsA1, present in the drug resistance region. The stability and transfer leading regions contain various important genes such as parAB, resD, ardA, psiAB, or ssb for plasmid maintenance, recombination and transfer reactions. When the genome of R64 was compared with those of other plasmids, varying levels of similarity were observed. It is suggested that genetic recombinations including the site-specific rfsF-ResD system have played an important role in diversity of genomes related to R64. It was found that R64 exhibits highly organized genome structure.

Characterization of Extended-Spectrum Cephalosporin–ResistantSalmonella entericaSerovar Heidelberg Isolated from Humans in the United States

During the past decade, extended-spectrum cephalosporin resistance has increased among human isolates of Salmonella enterica serovar Heidelberg, the fourth most common serotype in the United States. We therefore characterized 54 Heidelberg isolates with decreased susceptibility (minimum inhibitory concentrations >or=2 mg/L) to ceftriaxone or ceftiofur; 49 (90.7%) contained the CMY-type beta-lactamase (bla(CMY)) gene. The 49 bla(CMY)-positive human Heidelberg isolates demonstrated a high degree of relatedness; 4 clusters (25 isolates total) had indistinguishable XbaI and BlnI patterns by pulsed-field gel electrophoresis and were indistinguishable from 42 retail meat Heidelberg isolates. Further characterization of 15 of these isolates demonstrated that all of the bla genes were bla(CMY-2) and plasmid-encoded, and most (11/15) of the plasmids were approximately 100 kb in size and belong to the incompatibility group I1 (IncI1). All five IncI1 plasmids tested by plasmid multilocus sequence typing analysis were ST12. This report suggests that extended-spectrum cephalosporin resistance among human Heidelberg isolates is mediated by the spread of a common IncI1 bla(CMY-2) plasmid, which may have a preference for a particular genetic background.

Complete nucleotide sequences of plasmids pEK204, pEK499, and pEK516, encoding CTX-M enzymes in three major Escherichia coli lineages from the United Kingdom, all belonging to the international O25:H4-ST131 clone.

We determined the complete nucleotide sequences of three plasmids that encode CTX-M extended-spectrum beta-lactamases (ESBLs) in pulsed-field gel electrophoresis-defined United Kingdom variants (strains A, C, and D) of the internationally prevalent Escherichia coli O25:H4-ST131 clone. Plasmid pEK499 (strain A; 117,536 bp) was a fusion of type FII and FIA replicons and harbored the following 10 antibiotic resistance genes conferring resistance to eight antibiotic classes: bla(CTX-M-15), bla(OXA-1), bla(TEM-1,) aac6'-Ib-cr, mph(A), catB4, tet(A), and the integron-borne dfrA7, aadA5, and sulI genes. pEK516 (strain D; 64,471 bp) belonged to incompatibility group IncFII and carried seven antibiotic resistance genes: bla(CTX-M-15), bla(OXA-1), bla(TEM-1), aac6'-Ib-cr, catB4, and tet(A), all as in pEK499. It also carried aac3-IIa, conferring gentamicin resistance, and was highly related to pC15-1a, a plasmid encoding the CTX-M-15 enzyme in Canada. By contrast, pEK204 (strain C; 93,732 bp) belonged to incompatibility group IncI1 and carried only two resistance genes, bla(CTX-M-3) and bla(TEM-1). It probably arose by the transposition of Tn3 and ISEcp1-bla(CTX-M-3) elements into a pCOLIb-P9-like plasmid. We conclude that (i) United Kingdom variants of the successful E. coli ST131 clone have acquired different plasmids encoding CTX-M ESBLs on separate occasions, (ii) the bla(CTX-M-3) and bla(CTX-M-15) genes on pEK204 and pEK499/pEK516 represent separate escape events, and (iii) IncFII plasmids harboring bla(CTX-M-15) have played a crucial role in the global spread of CTX-M-15 ESBLs in E. coli.

Multilocus sequence typing of IncI1 plasmids carrying extended-spectrum β-lactamases in Escherichia coli and Salmonella of human and animal origin

Plasmids belonging to incompatibility group I1 (IncI1) are widespread in Enterobacteriaceae and are characterized by the presence of a cluster of genes encoding the type IV pili, contributing to the virulence of Shiga-toxigenic Escherichia coli. Recently, IncI1 plasmids were identified in E. coli and Salmonella strains of animal origin as responsible for the dissemination of beta-lactamase genes. Plasmid multilocus sequence typing (pMLST) was developed to discern naturally occurring IncI1 plasmids in homogeneous groups according to their allele assortment. pMLST was developed by selecting multiple target genes on the available complete IncI1 plasmid DNA sequences. Sixteen plasmids, all assigned to the IncI1 group by the PCR-based replicon typing method, were included in this study. They were analysed for beta-lactamase genes and typed by restriction fragment length polymorphism (RFLP) and pMLST. Sixteen plasmids identified in E. coli and Salmonella isolated from animals and humans in different countries carried bla(CMY-2), bla(CTX-M-15), bla(CTX-M-1), bla(CTX-M-14), bla(TEM-52), bla(SHV-12) or bla(TEM-1) beta-lactamase genes. These plasmids were classified by RFLP in nine different groups corresponding to the nine sequence types determined by pMLST. The pMLST method was suitable for rapid and easy subtyping of IncI1 plasmids. This study demonstrates that the pMLST method can contribute to the epidemiological description of circulation of specific resistance plasmids among beta-lactamase producers isolated from animals and humans.

Evidence for chromosomal and plasmid location of CMY-2 cephalosporinase gene in Salmonella serotype Typhimurium

Sir, Resistance to extended-spectrum cephalosporins has emerged among non-typhoid salmonellae (NTS) worldwide due to acquisition of plasmid-mediated b-lactamases including CMY-2. The isolation frequency of CMY-2-producing NTS in the USA has gradually increased since 1996 reflecting the widespread transmission of CMY-2-positive NTS among food animal reservoirs. The blaCMY-2 gene has been disseminated by plasmids to various Salmonella serotypes including Typhimurium. – 4 We report here on a human isolate of Salmonella serotype Typhimurium harbouring a CMY-2-encoding plasmid as well as a chromosomally located copy of blaCMY-2. Salmonella serovar Typhimurium AM19083 was submitted to the National Antimicrobial Resistance Monitoring System in 2003 from Massachusetts. Antimicrobial susceptibility was assessed by broth microdilution (Sensititre, Westlake, OH, USA). The isolate met the phenotypic criteria indicative of class C cephalosporinase production (i.e. resistance or decreased susceptibility to cefoxitin, ceftiofur and ceftriaxone). It was also resistant to ampicillin, amoxicillin/clavulanate and cefalotin, but susceptible to non-b-lactam drugs including streptomycin, sulphonamides, chloramphenicol, tetracycline, nalidixic acid and ciprofloxacin. AM19083 was positive in PCR assays with blaCMY-specific primers, and the sequence of the amplicon (369 bp) was homologous to an internal fragment of blaCMY-2 (from nt 271 to nt 639, in GenBank accession no. X91840). Isoelectric focusing of sonicated cell extracts in polyacrylamide gels containing ampholines showed production of a b-lactamase species with an isoelectric point of .8.4 corresponding to CMY-2. b-Lactam resistance was readily transferred by conjugation in broth cultures to a susceptible Escherichia coli host. The plasmid content analysis indicated transfer of an ~90 kb plasmid (designated p19083) that was positive in hybridization with a blaCMY-2 probe prepared as described previously. 5 Plasmid p19083 belonged to the incompatibility Group I1 (IncI1), as found by a PCR-based Inc/rep typing method. Furthermore, the PstI restriction profile of p19083 resembled restriction type B of the IncI1, CMY-2-encoding plasmids previously described in Salmonella Typhimurium and Salmonella Thompson isolates from the USA. A plasmid DNA preparation from AM19083 was electrophoresed through agarose gels (0.8%). DNA was then transferred to Hybond-N nylon membranes (Amersham Biosciences, Buckingham, UK) and hybridized with a digoxigenin-labelled blaCMY-2 probe. 5 These experiments showed not only the expected hybridization with plasmid p19083, but also a strong signal with the chromosomal material. This was not observed with preparations from two control strains of serotype Typhimurium, one of which (AM18447) also carried two CMY-2-encoding plasmids, p18447-S and p18447-L (~140 and 300 kb, respectively). To clarify this issue, an I-CeuI genomic analysis of isolates AM19083 and AM18447 was performed. The digested fragments were separated by PFGE (6.0 V/cm for 8 h/16 h with a pulsing time linearly ramped from 5 to 20 s/20 to 70 s) (Figure 1a). The blaCMY-2 probe was hybridized with a 16SrRNA-positive I-CeuI fragment of ~500 kb from isolate AM19083. Positive signals probably corresponding to the blaCMY-2-carrying plasmids were also seen (Figure 1b). Subsequent hybridization with Inc/rep probes showed that p19083 and p18447-S belonged to IncI1, whereas the blaCMY-2-carrying putative plasmid p18447-L and the 500 kb fragment from AM 19083 were not reactive (Figure 1c). In an attempt to rule out the existence of blaCMY-2-positive plasmids of high molecular weight in AM19083 and, also, to corroborate the plasmidic nature of p18447-L, the plasmid contents of AM19083 and AM18447 were analysed by PFGE after treatment of total DNA with S1 nuclease. The respective electrophoretic profiles indicated the absence of blaCMY-2-carrying ‘megaplasmids’ in AM19083, whereas a band of ~300 kb likely representing the linear form of p18447-L was observed in the preparations from AM18447 (data not shown). Finally, to further strengthen our hypothesis for the chromosomal acquisition of blaCMY-2 in AM19083, the relevant I-CeuI fragment was purified by electroelution. The resulting preparation contained blaCMY-2 as confirmed by PCR and sequencing. It was also negative in PCR assays designed for replicon typing of plasmids including IncI1 and IncA/C. Nevertheless, the possibility that plasmid segments including rep could have been removed during I-CeuI treatment cannot be excluded. Production of CMY-2 has been described in a wide variety of NTS serotypes in the USA. The plasmidic location of blaCMY-2 is critical for spread of this resistant determinant. The present findings confirm the prominent role of IncI1 plasmids in this process. Additionally, the likely chromosomal carriage of blaCMY-2 underscores the operation of efficient mobilization mechanisms commonly involving ISEcp1 and/or IS26. Multiple copies of a resistance gene may increase the antimicrobial resistance levels and provide more of a selective advantage. Expression of the chromosomal CMY-2 and its possible contribution to b-lactam resistance levels were not studied. b-Lactam MICs for Salmonella Typhimurium AM19083, however, were comparable with those observed for other Salmonella strains carrying only plasmidic blaCMY-2, such as AM18447, but, for many of Journal of Antimicrobial Chemotherapy (2008) 61, 1389–1399 doi:10.1093/jac/dkn116 Advance Access publication 18 March 2008